Obesity and insulin resistance is associated with alterations in innate immune function. Phenotypic alterations in adaptive immune cells (T cells) play a crucial role in the macrophage (innate) inflammatory response. In this proposal, we hypothesize that depletion of T regulatory cells (Treg) in visceral adipose tissue is a key defect that worsens insulin resistance. We propose to test this hypothesis in a novel transgenic mouse model (Foxp3gfp.KI) that expresses green fluorescent protein (GFP) under control of a Foxp3 promoter allowing studies on Treg function in conjunction with studies of Treg and innate immune cells in visceral adipose of humans with obesity. In Aim 1, we will investigate the relative proportion and determinants of Treg in obese Foxp3gfp.KI mice (C57/BL6 strain) and human subjects. Our hypothesis being that adipose tissue Treg cells are important predictors of insulin resistance and inversely correlate with the extent of adipose inflammation. In Aim 2, we will investigate the hypothesis that in vitro differentiation of Foxp3+ T cells is critically dependent on the activation state of the adipose dendritic cells (ATD, F4/80+CD11c+) with pro-inflammatory ATD prevalent in visceral adipose of obese mice initiating less Treg differentiation than ATD in lean mice. We will investigate the role of cell contact vs. cytokine influences on Treg differentiation. In the third specific aim, we will explore the impact of Treg depletion in insulin resistance and adipose inflammation, Foxp3-GFP (C57BL/6 background) mice will undergo daily injections of antibodies against CD25 in order to deplete Tregs for a duration of 4 weeks. The mice will be randomized to a standard chow diet or high fat diet for 10 weeks. Whole body insulin sensitivity, adipose inflammation and M1/M2 phenotype of ATM/ATD population will be determined in the two groups. Our hypothesis is that Treg depletion exaggerates a pro-inflammatory phenotype in visceral adipose tissue and insulin resistance in response to diet induced obesity. Using a novel susceptible model that allows unprecedented abilities to track Treg function with confirmatory studies in human adipose samples, this proposal offers an unprecedented opportunity to elucidate relevant mechanisms responsible for the pathogenesis of insulin resistance and inflammation in obesity. PUBLIC HEALTH RELEVANCE: Obesity and metabolic complications relating to obesity are spreading at epidemic proportions globally. It is now recognized that these disorders are characterized by major changes in cellular immune function. Understanding how the immune system affects susceptibility and promotes metabolic complications is central to prevention and treatment. In this study, we will perform studies in an animal model of obesity that will allow us unprecedented ability to track specific immune cells called T regulatory cells and assess how these cells affect the function of other immune cells such as effector T cells and macrophages. We will additionally perform confirmatory studies in humans with obesity to ensure applicability of findings to human disease.